Kinetic Characterization of Kbc1 Acetoacetate Utilization

Kbc1 activity was detected using our assay previously described for Acs1 activity except sodium acetoacetate was used in place of sodium acetate (18 (link)). Michaelis-Menten constants were determined for the acetoacetate, CoA, and ATP substrates by first determining the optimal concentrations of these substrates so that they would be in excess without, in the case of CoA, being high enough to inhibit the reaction. Substrates provided in excess allow the apparent Km to closely approximate the actual Km. The EnzChek Pyrophosphate assay kit (Thermo) was used with reagents prepared by manufacturer’s standards, and with 4 mM MgCl₂, 10 mM DTT, 4.5 μg/mL Kbc1, 100 μM CoA, and 200 μM ATP per 50 μL reaction. The reagents were mixed and aliquoted at room temperature, allowed to incubate for 15 min at 37°C to mop background phosphate contamination, then acetoacetate (final concentration of 1 mM) was added to the reaction, and the plate was read continuously for 40 min at 37°C in a SpectraMax i3X Multi-Mode plate reader (Molecular Devices) at absorbance 360 nm. To test possible substrates, a dilution series of propionate, butyrate, 3-hydroxybutyrate, or acetate was added in place of acetoacetate.